Capillary electrophoresis (CE) has developed into a powerful analytical method due to its high resolution and low volume requirement. Providing fast and efficient separations, CE offers significant advantages over conventional slab gel electrophoresis. However, a major problem encountered in CE application is the interaction of analytes, such as proteins, with exposed surface silanol groups on the capillary wall. This interaction results in a loss of efficiency and irreproducible binding. The electrophoretic mobility of analyte in CE is a constant value depending on the analyte's characteristics, buffer composition and temperature. A second mode for the analyte motion is driven by electroosmotic flow (EOF) which originates from the negatively charged silica capillary surface. The apparent mobility of an analyte is determined by the combined electrophoretic mobility and the EOF. Due to the variation of EOF from run-to-run and the non-specific interaction of the analyte with the inner surface of the capillary, it is very difficult to achieve reproducible separations. A number of CE applications, such as protein separations, DNA sequencing and peptide mapping, require reproducible separation, such as consistent retention time and high efficiency, as well as minimum non-specific bindings. Therefore a solution for suppressing EOF and minimizing non-specific interactions is very important for CE's utilities.
Surface coating is a natural step for controlling EOF and the non-specific interactions. In the past decade, several different methods have been developed to coat the capillary inner wall surface. Typical ones include physically adsorbed coating, i.e. dynamic coating, and chemical bonded coating, i.e. permanent coating. A number of different dynamic coatings produced by adsorption of polymers from an aqueous solution have been described. (Madabhushi, Electrophoresis, 19, 224, 1998; Chiari, et al. J. Chromatogr. A 817, 15, 1998; Gilges, et al. J. High Resolution Chromatography 15, 452, 1992). Whereas, a common problem of these coatings is that the polymers can be easily removed from the capillary wall simply by washing with water or buffer solution. Therefore, unless otherwise stabilized, these coating are useful in reducing EOF only when a small amount of polymer is dissolved in the running buffer and can replace the polymer removed by running buffer from the surface. U.S. Pat. No. 6,410,668 (Chiari et al. “Robust Polymer Coatings”) described another example of dynamic coating for improvement of separation by using uncharged water-soluble polymers that could reduce EOF in capillary electrophoresis temporally without addition of any polymer to the running buffer. The polymer coating was achieved by a thermal treatment of an immobilized PVA on the capillary wall. This coated capillary gave a low EOF up to pH 9. However, only 40 runs were possible at pH 8.5 without loss of efficiency. Overall, dynamic coatings are physically attached to the capillary surface and can be easily removed. The detached polymer will contaminate the analyte. Moreover, the dynamic coating is a very thin polymer layer, usually less than 1 nm in thickness. It is not effective in reducing non-specific interaction with charged molecules, especially proteins.
A chemical bonded coating is, in general, more stable than a physically adsorbed coating. Several articles have summarized chemically modified capillaries that were designed to minimize the presence of surface silanols and reduce analyte interactions (Dolnik et al. J. Biochem. Biophys. Methods 1999, 41, 103-119; Chiari et al. Anal. Chem. 1996, 68, 2731). These modifications involve attaching or creating a polymeric layer on the surface of the capillary through various coupling chemistries. Hjerten (J. Chromatogr. 347, 191, 1985) showed a two-step coating process by attaching a bifunctional silane on the surface of the capillary followed by in situ free radical polymerization of a vinyl group containing monomer, e.g. acrylamide. The presence of a polymerizable C═C group was essential in both the monomer and silane for coupling. Cobb et al. (Anal. Chem. 1990, 62, 2478) used Grignard reaction to link a vinyl group (—CH═CH2) directly to the capillary surface, followed by the same free radical polymerization of acrylamide as Hjerten method. U.S. Pat. No. 6,372,353 (Karger et al. “Coated surface composing a polyvinyl alcohol (PVA) based covalently bonded stable hydrophilic coating”) disclosed a PVA coating by free radical polymerization of vinyl acetate, followed by hydrolysis. U.S. Pat. No. 5,792,331 (Srinivasan et al. “Perform polymer coating process and product”) described a coating method for covalently bonding a coupling agent having capability of forming a free radical under hydrogen abstraction conditions and then contacting with polymers at elevated temperature to form a coating layer on the surface. Strege et al (J. Chromatography 665, 63, 1993) reported in-situ cross-linking polymerization of polyacrylamide on capillary surface for separation of basic proteins with no added cationic additives in the buffer. Recently, Wan et al (J. Chromatography A 924, 59, 2001) reported polymerization of dimethylacrylamide at low concentration in the presence of isopropyl alcohol to achieve high separation efficiency. All above methods utilized free radical polymerization. For free radical polymerization, the radicals are formed in the solution as well as on the surface. With majority of the polymers formed in the solution, this coating method results in no control of polymer molecular structure, coating uniformity and thickness. These important properties have great influence on the EOF and non-specific interactions. A big disadvantage of using this method is that the free polymer could precipitate from the solution, which may block micro-channel of capillary, or the polymer chains formed in the solution could cross-link the capillary walls to clog the capillary. Such an uncontrolled process makes the coating irreproducible, and introducing variations in separations. It makes production difficult. There are strong interest and need in industry and academia for making stable, reproducible and uniform polymeric coatings on the inner wall of the silica capillary by a simpler method. For example, multiple capillary array technology, which needs to handle 96 or more capillaries at the same time, requires simple and reliable coating procedures. With a controlled process to make a polymer coating, various polymeric structures of the coatings can be manipulated to achieve better separation results. Moreover, the polymer coated support surfaces can be used for separating the components in a fluid stream such as micro-channel separation or liquid chromatography.